Identification of proteolytic activities in the cytosolic compartment of mature human erythrocytes.

Abstract

Individual lysates from human erythrocyte suspensions, completely deprived of leucocytes and were assayed for a number of proteolytic activities using both naturally occurring and synthetic substrates. Removal of hemoglobin by batchwise DEAE-cellulose chromatography did not modify the complement of the various proteolytic activities which were then fractionated by means of chromatography on a column of DEAE-cellulose, followed by conventional techniques such as gel chromatography and preparative electrophoresis. This procedure allowed a number of proteinases to be identified in the erythrocyte cytosol while providing a tool for their selective though partial separation. The following peptidases were found to be present in the soluble fraction of mature human erythrocytes: (a) a neutral endopeptidase having an approximate molecular weight of 110 000; (b) three acidic endopeptidases, with pH optima between 2.5 and 3.5, showing molecular and functional properties almost identical with those of the three proteinases previously purified from solubilized erythrocyte membranes [Pontremoli et al. (1979) Biochem. J. 181, 559--568]; (c) two dipeptidylaminopeptidases whose molecular weights are around 80 000 and tentatively identified as dipeptidyl aminopeptidases II and III, respectively, on the basis of their substrate specificities and pH optima; (d) presumably two aminopeptidases, having an approximate molecular weight of 80 000 and classified as an aminopeptidase with broad substrate specificity and an aminopeptidase B, respectively. No evidence for any carboxypeptidase activity was found in the cytosolic compartment of mature human erythrocytes.